Increase in prevalence of Streptococcus pneumoniae serogroup 24 in children upon introducing 13-valent pneumococcal conjugate vaccine in Japan

After introducing the 13-valent pneumococcal conjugate vaccine (PCV13) for children, a change in the prevalence of different Streptococcus pneumoniae serotypes that cause invasive pneumococcal diseases (IPDs) has been observed. The prevalence of vaccine serotypes has decreased and that of non-vaccine serotypes has increased. Currently, serogroup 24 has become one of the major non-vaccine serotypes causing IPDs in children in Japan. The aim of this study was to characterize clinical and genomic features of S. pneumoniae serogroup 24 strains isolated from sterile body sites in Japanese children. Serotyping, multi-locus sequence typing and genomic analysis of capsular polysaccharides of 61 strains of serogroup 24 were performed from 2015 to 2021. Among the 61 strains, 36, 23 and two belonged to serotypes 24F, 24B and 24C, respectively. The 24F sequence type (ST) 2572 and 24B ST 2572 were the major serotypes and sequence types observed from 2015 to 2019. By contrast, 24F ST 162 and 24B ST 2754 were the two major serotypes and sequence types observed after 2020. Two strains of serotype 24C were detected for the first time in Japan. Sequence analysis of the abpA gene, which plays a role in the synthesis of capsular polysaccharides in S. pneumoniae , was performed to distinguish different strains of serogroup 24. After the introduction of PCV13 in Japan, serogroup 24 has become one of the most prevalent non-vaccine serotypes causing IPDs in children. This serogroup has not been targeted in the next-generation pneumococcal conjugate vaccines. Therefore, monitoring of S. pneumoniae serogroup 24 that causes IPDs in children is essential.


INTRODUCTION
Streptococcus pneumoniae is an alpha-haemolytic bacterium. In previous studies, microscopic examination of the pneumococcus revealed that it is a Gram-positive diplococcus, and a majority of S. pneumoniae are surrounded by a capsule. Currently, more than 100 capsular serotypes of S. pneumoniae are known [1]. S. pneumoniae is one of the leading causes of invasive bacterial diseases such as meningitis, bacteraemia and bacteraemic pneumonia [2,3]. Pneumococcal conjugate vaccines [PCVs; heptavalent (PCV7), 10-valent (PCV10) and 13-valent (PCV13)] were developed to prevent invasive pneumococcal diseases (IPDs) in children.
Recently, next-generation PCVs, namely 15-valent and 20-valent PCVs, were approved for prevention of IPDs in adults in the USA [4,5]. These next-generation PCVs are expected to be introduced for children. After the introduction of PCVs for children, there OPEN ACCESS has been a pronounced decrease in cases of IPDs caused by serotypes that are targeted by the PCVs [vaccine serotypes (VTs)] in many countries. However, the prevalence of non-vaccine serotypes (NVTs) of S. pneumoniae that cause IPDs has increased [6].
Serogroup 24 includes serotypes 24F, 24A, 24B and the newly identified 24C [7]. Serotype 24F is one of the most prevalent NVTs causing IPDs [8,9]. The prevalence of serotype 24 differs among countries. A systematic review of serotype distribution of paediatric IPDs in the post-PCV era revealed that 24F is prevalent in Europe and the Western Pacific region, but not in North America [8]. Previously, it has been rarely isolated from children with otitis media and pneumonia in Japan, which suggests an increase in the invasiveness of this serotype [9]. A time series analysis conducted as part of a national survey in France demonstrated a sharp increase in the number of pneumococcal meningitis cases in children, which were primarily related to serotype 24F [10]. This phenomenon was observed between 2012 and 2014 after the introduction of PCV13. Although pneumococcal epidemiological change is influenced by not only vaccine pressure but also other internal and external factors, implementing a highly reactive surveillance system in each country is necessary to verify the local serotypic appropriateness of new-generation PCVs [10]. In Japan, PCV7 was introduced in February 2010, and administration of PCV13 as a routine vaccine began in November 2013. A survey of IPDs in children showed that the incidence of IPDs caused by serotype 24F increased considerably after the introduction of PCV13. In addition, we also found that the incidence of IPDs caused by serogroup 24 strains in children increased significantly after the introduction of PCV13 in Chiba Prefecture, Japan, while cases of IPD caused by serotype 24B strains have also increased since 2019 [11]. Therefore, a detailed analysis of serogroup 24 strains isolated from patients with IPD is required. Herein, we report the molecular analysis of S. pneumoniae serogroup 24 strains isolated from children after the introduction of PCV13 in Japan, mainly in Chiba Prefecture. In particular, we aimed to determine the genetic basis of structural differences in the capsules of pneumococci belonging to serotypes 24F, 24B and 24C.

Pneumococcal isolates
We collected S. pneumoniae strains from the cerebrospinal fluid or blood of paediatric patients with IPDs aged less than 15 years with IPDs. All these patients were admitted to various hospitals in Japan between January 2015 and December 2021. We collected samples from all children and adolescents with IPDs in Chiba Prefecture. Chiba is one of the 47 prefectures in Japan, located next to Tokyo. It has a population of 6.3 million, which accounts for approximately 5 % of the total population of Japan. Samples from nine other prefectures were obtained from clinicians on request, because of the lack of an active IPD surveillance system including bacterial analysis in the nine prefectures. Overall, we obtained 61 strains of S. pneumoniae serogroup 24 from sterile body sites of children with IPDs from various regions in Japan, covering 10 of the 47 prefectures. An IPD case was defined as the occurrence of S. pneumoniae in cerebrospinal fluid, blood or other normally sterile body sites.

Preparation of chromosomal DNA
Bacterial strains were incubated overnight on blood agar medium supplemented with 5 % sheep blood at 35 °C in 5 % CO 2 . The colonies were then inoculated in 10 ml of Todd-Hewitt broth (THY) supplemented with 0.5 % yeast extract and grown to the mid-log phase. After incubation, the culture broth was centrifuged and the precipitates were extracted for chromosomal DNA isolation from each strain using MORA EXTRACT (Kyowa Hakko Industries). The concentration of chromosomal DNA was measured using a NanoDrop One (Thermo Fisher Scientific), and the concentration was adjusted to 100 ng µl -1 . This genomic DNA was used for the PCR assay.

PCR conditions
All PCR assays were performed with the same programme and reagent concentrations: 2× KOD One PCR Master Mix (TOYOBO), 0.3 μΜ of each primer and 0.5 µl DNA template mixed to a total volume of 25 µl. PCR was performed under the following conditions: initial denaturation at 98 °C for 1 min, followed by 30 cycles at 98 °C for 10 s, 55 °C for 5 s and 68 °C for 5-20 s, in a T-100 Thermal Cycler (Bio-Rad). Amplification was confirmed using electrophoresis of 3 µl of the PCR products in 1.5 % (w/v) agarose gels and visualized by staining with ethidium bromide.

Sanger sequencing
The PCR products were cleaned using the FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and used as samples for Sanger sequencing. Sanger sequencing of the PCR products purified on an ABI PRISM 3130xl Genetic Analyzer (Thermo Fisher Scientific) was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit, at the Medical Mycology Research Center of Chiba University.

Identification of S. pneumoniae serogroup 24 strains
All isolated strains were cultured on blood agar plates supplemented with 5 % sheep blood (Becton Dickinson) and incubated overnight at 37°C under 5 % CO 2 . Optochin susceptibility testing was performed using the disc diffusion method. The strains were streaked on sheep blood agar medium, and discs containing 5 µg optochin (Eiken Chemical) were placed on the blood agar medium and incubated overnight at 35 °C under 5 % CO 2 . According to the manufacturer's instructions, the strains were identified as S. pneumoniae by confirming susceptibility with an inhibition zone of ≥14 mm. PCR assays targeting the lytA gene were performed to verify that the isolated strains belonged to S. pneumoniae. The isolates were serotyped by performing Quellung reactions using pneumococcal antisera (Statens Serum Institute).

Molecular characterization of the gene encoding capsular polysaccharides (cps)
For the 61 strains, the cps gene of strain ST 2572 was analysed using PCR and DNA sequencing. For the PCRs, primers FI3 (5′-TCTT AGTT CCAT GGGA TGCT TTCT GTGTG-3′) and FI4 (5′-CGCT GAAC TTTT GTAG TTGC TGTC TGGTCAAC-3′), and original primers were used [13,14]. In S. pneumoniae, cps clusters (responsible for capsular biosynthesis) are generally located between dexB and aliA loci; thus, the primers FI3 and FI4 were designed using the sequences of these two flanking genes, to ensure that the intervening region containing the complete cps sequences could be amplified. PCRs were also performed with primers designed using the known sequences of genes in the cps locus of serogroup 24. The original primers were created using primer design software (Primer-blast) based on the sequences available on GenBank, accession numbers CR931688 (serotype 24F) and CR931687 (serotype 24B), which are known sequences at the cps locus of serogroup 24. The PCR products of the cps locus were sequenced using the Sanger method.

Relationship between abpA genes of the cps loci and ST/serotype combinations
The cps sequences obtained in this experiment were aligned and compared with the reference cps loci cps24F (CR931688) and cps24B (CR931687) [15]. The abpA gene present in the cps locus was analysed in the 61 strains using PCR and DNA sequencing. Primers 8582F (5′-CAGC TGGA AAGT TAAT GGTTGGT-3′) and 9758R (5′-ACCA ATCA AACC AGAA GCTCCA-3′) were used in the PCR, following a published protocol [15]. Annotation of the determined gene sequences showed that the cps locus configuration of all serogroup 24 ST 2572 strains was the same as that of the gene used as a reference (CR931688). Sequence comparison revealed some scattered single nucleotide polymorphisms (SNPs) at the cps locus but no mutations common to strains 24F ST 2572 and 24B ST 2572 (except the abpA gene sequences). Therefore, we analysed the abpA gene sequences of isolates 24F, 24B and 24C that were isolated in this study. The PCR products were sequenced and the nucleotide sequence of the abpA gene was determined. The relationship between the abpA genes present in the cps locus and the ST/serotype combinations of the 61 strains was examined.

Identification of S. pneumoniae serogroup 24 strains
The results of optochin susceptibility tests, PCR assays targeting the lytA gene and Quellung reactions using pneumococcal antisera confirmed that all 61 strains belonged to S. pneumoniae serogroup 24. Serogroup 24 was isolated in 31 of the 158 cases in Chiba Prefecture, and in 30 of the 112 cases in the other nine prefectures, which accounted for 19.6 and 26.8 % of all identified cases, respectively. A comparison of the sampling years, background of the patients and serotype/ST of the strains between Chiba and other prefectures is presented in Table S1 (available in the online version of this article). All 61 cases involved children aged 8 years or younger, with the majority of them <2 years of age.

Relationship between abpA genes of the cps loci and ST/serotype combinations
The blast search results revealed that the cps loci (except the abpA gene sequences) of all serogroup 24 strains shared 100 % identity. The cps loci (except the abpA gene sequences) of all strains of 24F ST 2572 and 24B ST 2572 were identical. Therefore, we analysed the abpA gene sequences in strains 24F, 24B and 24C that were isolated in this study. The abpA genes of the 24F ST 2572 strain had identical nucleotide sequences. Fig. 2 shows the relationship between abpA sequences and ST/ serotype combinations; the analysis was performed using the abpA gene sequence of 24F ST 2572 strain as the reference sequence. The abpA gene sequence was used to distinguish among 24F, 24B and 24C strains. The C-terminal sequence of the abpA gene was different between the two 24C strains. All 24B ST 2754 strains had an identical single nucleotide deletion in the abpA gene sequence, causing a frameshift. On the other hand, each of the nine 24B ST 2572 strains had a different point mutation in the abpA gene, causing an amino acid substitution. We also found that ST162 had differences shared across all ST162 isolates and these were not present in other STs. We observed diversity within ST 2572 serotype 24B, while the recently emerged serotype 24C was identified across at least two STs.

DISCUSSION
After the introduction of PCVs, the incidence of IPDs caused by VTs has decreased dramatically in many countries. On the other hand, the incidence of IPDs caused by NVTs has increased [6,16]. Among all the NVTs, serogroup 24 was one of the most prevalent serotypes in Japan [11]. Surprisingly, serogroup 24 is a major serogroup causing IPDs in children, but it has been rarely isolated from adults with IPDs in Japan [17,18]. This has also been reported in France [19]. However, in Denmark, the IPD caused by serotype 24F has increased not only in children but also adults [20]. Serogroup 24 is categorized into four serotypes, namely 24F, 24A, 24B and 24C. Among them, serotype 24C has been recently identified [7]. In this study, we analysed the relationships between serotypes and ST combinations of several serogroup 24 strains and their clinical manifestations.
In this study, serotypes 24F, 24B and 24C were isolated from children with IPDs. Serotype  . In our study, all 61 strains were susceptible to penicillin G, whereas 24F ST 162 strains were cotrimoxazole-resistant. In the future, it will be essential to closely monitor the trends associated with serotype 24F ST 162.
Interestingly, the major serotype changed from 24F to 24B during the study period, especially serotype 24B ST 2754, the prevalence of which increased rapidly in 2021. Most of the serotype 24B ST 2754 strains were isolated from children <2 years of age with bacteraemia or bacteremic pneumonia. Serotype 24B ST 2754 has not been reported from other countries. These findings suggested that this phenomenon was caused by the clonal spread of serotype 24B ST 2754 in Japan, similar to the one observed in the case of serotype 12F ST 4846 [23]. However, to clarify the origin of 24B ST2754, further studies are needed. This is the first report on the isolation of serotype 24C from patients with IPD in Japan. In this study, only two serotype 24C strains were identified, the first of which was isolated in 2019. These two 24C strains had different STs. Serotype 24C ST 2572 was isolated from a patient with meningitis. Until now, serotype 24C has been isolated from patients in Germany and England [7]. Our findings suggest that serotype 24C has spread to various regions of the world.
Previously, variations in the sequences of the abpA, abpB and wcxG genes were the reason for the differences observed in the subtypes of serogroup 24 [15]. However, a recent study has revealed that these sequence variations are not serotype-specific. Ganaie et al. [7] analysed the sequence of an approximately 2.6 kb region of the cps loci (including the wcxG, abpA and abpB genes) of several strains representing the four related serotypes 24F, 24A, 24B and 24C. Gene sequences of wcxG and abpB in these four serotypes were similar to the published sequences, and no serotype-specific differences were observed. By contrast, the abpA sequences were not identical among all four serotypes [7]. Therefore, we analysed the abpA gene sequences in serogroup 24 strains isolated in this study. The abpA gene, formerly known as abp1, is 720 bp long and encodes a transferase enzyme (240 aa) that is involved in the biosynthesis of CDP-arabinitol [24,25]. In our study, all 18 strains of serotype 24F ST 2572 had the same amino acid substitutions in the sequence of the protein encoded by the abpA gene. By contrast, there is considerable diversity within serotype 24B ST 2572. Analysis of the abpA gene sequence helped distinguish between 24F and 24B types of the ST 2572 strain. The C-terminus of the abpA gene in both 24C strains was different from that of the 24F ST 2572 strain. This change in The genetic variability of serotypes 24F, 24B and 24C is an interesting phenomenon. One possibility is that serotype 24F strains initially colonized the nasopharynx, and then human-to-human transmission occurred. During this invasion process of different body sites, the 24F serotype mutated to either the 24B or 24C strain [26]. In other words, when the 24F strain invaded deeper tissues, it might have encountered the host immune response that targeted arabinitol. Thus, the 24F serotype switched to the 24B or the 24C serotype to avoid an immune response to the host [27]. Another possibility is that immunologically related pneumococcal capsular serotypes were concomitantly present in the same patient. The simultaneous recovery of two serotypes of serogroup 24 from exudates of the middle ear and blood culture samples has been reported [28]. However, these proposed mechanisms underlying the variations have not been fully demonstrated. Considering the above hypothesis, a thorough investigation of serogroup 24 strains isolated from sterile body sites and the nasopharynx is necessary. Further studies are needed to clarify the precise mechanisms involved.
This study has several limitations. First, a nationwide survey was not conducted and the regional differences in Japan were not considered in this analysis. Although sampling from Chiba Prefecture was population-based, requested samples from the other nine prefectures may have been biased. Second, the sampling period included the time after the introduction of PCV13. We investigated MLST of 11 serogroup 24 strains isolated during 2010-2014. Among them, eight strains belonged to serotype 24F ST 5496, and one strain each belonged to serotype 24F ST 2572, serotype 24F ST 9609 (one locus mismatch of ST 2572) and serotype 24B ST 2572. However, we could not perform a precise analysis of the strains before the introduction of PCV13. Finally, we could not conduct whole genome sequencing of the strains nor analyse the factors responsible for the invasiveness of the isolated strains.
In conclusion, we analysed S. pneumoniae serogroup 24 strains isolated from children with IPDs in Japan. This study was conducted after the introduction of PCV13 in Japan. Serotype 24F ST 2572 was the major serotype causing IPDs from 2017 to 2019. By contrast, 24F ST 162 and 24B ST 2754 were the two major serotypes observed after 2020. Two strains of serotype 24C were collected during the study period. Analysis of the abpA genes present in cps loci helped in distinguishing between different strains of serogroup 24. After the introduction of PCV13 in Japan, serogroup 24 is one of the most prevalent serogroups causing IPDs in children. This serogroup is not targeted by the next generation of PCVs, namely 15-valent and 20-valent PCVs. We need to closely monitor the clinical features associated with serogroup 24 in the future.  Editor's comments Furthermore, given the open access policy of Access Microbiology, I would kindly ask you to consider the possibility to make the data that support this study available on a public database.

Response
We agree with your suggestion. The data that support the findings of this study are available on a public database.

Editorial Office requirements
Please upload figures as separate, high resolution, editable files.Acceptable file types arePDF, GIF, TIFF, EPS, JPEG, PNG, SVG, and PPT. Please ensure the legendsare in the mainmanuscript.

Response
We have uploaded the figures (PDF) as separate files. The figure legends have been compiled at the end of the manuscript.

Reviewer 1's Comments to the Authors
Methodological rigour, reproducibility and availability of underlyingdata. The methods used are generally ok. However, there are several gaps inthe methodology descriptions that would make reproduction difficultand therefore need to be addressed.

Response
We thank you for your comments. We have made the necessary revisions in the manuscript accordingly. 1. The age distribution of cases is not clear. I would argue that 15-year-olds are better described as adolescents and not children and wouldurge the authors to reflect on this and alter.

Response 1
We thank you for your comment. In terms of age distribution of the cases, all individuals who suffered from IPD caused by serogroup 24 were under 8 years of age. We have revised the relevant sentence in the methods section and added a description in the results section. 2.For those unfamiliar with the study locations, it would be good togive a brief overview of the prefecture in terms of location andpopulation, and the sites these isolates came from.

Response 2
We have added detailed information of Chiba Prefecture, which is the main study site.
"Chiba is one of the 47 prefectures in Japan, located next to Tokyo. It has a population of 6.3 million, which accounts for approximately 5% of the total population of Japan. " 4.You should indicate how many pneumococcal isolates were available intotal from which these 61 were taken. That would give an indication ofthe overall prevalence of serogroup 24 as a cause of IPD relative toother serotypes.

Response 4
We thank you for your important comment. We have added the number of tested strains. 6.You also need to give the PCR reaction conditions (primerconcentrations, mastermix, thermocycler used and cycling conditions).This also applies to cps sequencing (where you should also supply thename of the sequencing instrument used and where it was done.

Response 6
We have described the detailed PCR and cpssequencing method used in this study.

Response 8
For clarity, we revised the sentence as follows.
"The results of the optochin susceptibility tests, the PCR assays targeting the lytAgene, and Quellung reactions using pneumococcal antisera confirmed that all 61 strains belonged to the S. pneumoniaeserogroup 24. " (page 4 Line 155-157) Question 9 2.Line 144. Please can the authors clarify how in the methods it statesthat 61 serogroup isolates were acquired and here you state that it was31, is this just a typo?

Response 9
We thank you for your comment. Among all strains, 31 strains were isolated in Chiba Prefecture. We have made the necessary revisions throughout the manuscript.  Figure 1. I do not have an issue with presenting counts however youshould also present proportion as this figure does not illustratewhether overall IPD has remained constant over this period. Also, Y axisneeds to be clearer.

Response 10
We thank you for your comment. We have added the number of overall IPD cases in each year. The y-axis represents the number of serogroup 24 strains.
( Figure 1 and the

Response 12
We analyzed all strains and revised the relevant sentence to address the concern. (page 4 Line 183-185) Question 13 6.Please put the detail that all 24F ST2572 abpA were identical intoTable 2.

Response 13
We created Figure 2, instead of Table 2 and presented the detailed data in Figure 2.  Table 2 would be better communicatedalongside a Figure. Response 14 We thank you for your suggestion. We created Figure 2 to replace Table 2. Literature analysis or discussion 1. I don't believe ST2754 has been previously associated with 24B and Ithink this potential capsule switch requires further discussion.

Response 15
We agree with your comment. We have added the following sentence in the manuscript. 5. The 'limitations' section of the study requires expanding. AMR datawould have been useful, and achievable. Whilst more challenging andcostly the lack of genomic resolution of lineages, i.e., GPSCs, doesmake contextualisation difficult.

Response 19
We thank you for your comments. We have added the AMR data in the discussion section. In this study, we could not analyze the whole genome sequence of the strains;this point has been emphasized as a limitation. Line 90: There is more to this reference than is given in thisintroduction. For the reader it would be useful to expand to highlightthe GPSC narrative of these findings.

Response 23
We thank you for your comment. We have added more information from the cited study in the Introduction. Line 96-97: This implies that this has not been described previously,whereas it has. Perhaps rephrase to indicate this is a description ofisolates from a specific geographic area.

Response 24
We have revised the sentence considering your comment. (

Response 25
We thank you for the suggestion. We have cited the study (reference [12]). We have fixed the mistake and revised the text throughout. We apologize for the oversight.

Others
We have added new references (Ref. 12, 19) considering the reviewers' suggestion and the in-text citations have been renumbered accordingly.

Question 1
Methodologically this is mainly simple and clearly explained.
My main methodological questions are about the sampling. The Chiba sampleseems to be complete. I am less sure of the extent to which the widersurvey of samples across Japan is representative or may be biased. Thiscould be in terms of differential sampling across the time period, theselected clinical -demographic population, and subtype. Here thedescription "samples from other prefectures were obtained fromclinicians on request" does not give much idea. It would be good to bemore explicit on this and the extent to which this may have biased thepattern across years, subtype or patient demography or clinical picture.If this is likely then features such as counts over time may be betterrestricted to the Chiba prefecture sample or at least for this to bevisible in presentation and discussion of results across thesedimensions.

Response 1
We thank you for your valuable comments. We have expanded the sampling details in the manuscript. We have also added the supplemental table comparing the samples from Chiba prefecture with those from other nine prefectures. We have also highlighted this point in the limitations.
"The samples from nine other prefectures were obtained from clinicians on request, because of the lack of active IPD surveillance system including bacterial analysis in the nine prefectures. " The other sampling issue is the overall time frame. The title "Increasein prevalence of Streptococcus pneumoniaeserogroup 24 in children uponintroducing13-valent pneumococcal conjugate vaccine in Japan" would leadme to expect as ample comparing afterwards with before. However, thevaccination was introduced in 2010 (7 valent) and 2013 (13 valent) butthe sample extends forward from 2015. At least for the 7 valent this wasvery late in terms of the main carriage group for most S. pneumoniaebeing your children with quite extensive coverage in place for thispopulation before 2015.The rationale for this sampling period vs whatmight be expected and inferred from the title is not discussed ormentioned in limitations.

Response 2
We thank you for your valuable comment. We investigated MLST of 11 serogroup 24 strains isolated during 2010-2014. Among them, there were eight strains of serotype 24F ST 5496, and one strain each of serotype 24F ST 2572, serotype 24F ST 9609 (one locus mismatch of ST2572), and serotype 24B ST 2572. We have added the dataand referred to the sampling issue of the time frame in the limitations.
"In addition, we also found that the incidence of IPDs caused by serogroup 24 strains in children significantly increased after the introduction of PCV13, in Chiba Prefecture, Japan , " The data summary statement notes that "No data was generated during thisresearch." This seems to conflict with a description of methods togenerate data and the data then presented in results as part of thisresearch. There is also no statement on depositing of the data generated. Part of the usefulness of this research is for these data to be curatedand accessible to other researchers and this should be assured anddescribed.

Response 3
We thank you for your comment. We agree with your suggestion. The data are presented in the results section. Our data are curated and accessible to other researchers.
We have revised the data summary statement. (Data summary statement) Question 4

Presentation of results
The paper generally presents data clearly. In comparing the sequences intable 2 the main findings to me look to be that (i) ST162 has manydifferences shared across all ST162 isolates and not present in theother STs., (ii) there is a lot of diversity within ST2572 serotype 24B,the recently emerged (or at least identified) serotype 24C is presentacross at least sequence types. I think that it would be useful to pullout these themes in the results descriptions and discussion.

Response 4
We thank you for your valuable comment. We have revised the results and discussion section accordingly.
"We also found that ST162 had differences shared across all ST162 isolates and were not present in other STs. We observed diversity within ST2572 serotype 24B, while the recently emerged serotype 24C was identified across at least sequence types. " Also (Line237 : "…point mutations were detected in the abpA sequences of all the24B ST 2754 strains,…") sounds to me as though describing a range ofmutations but Table 2 appears to show a single shared difference across24B ST 5724 vs the reference sequence used in the paper.

Response 5
We thank you for your comment. We apologize for the confusion regarding the data of 24B ST 2754 strains.
We created Figure 2, instead of Table 2, and presented the detailed data of 24B ST5724 in Figure 2. As above -there may be a reason to present the Chiba prefecture resultsas a population sample and other isolates not as a representativepopulation sample subject to how this process of sampling more widelywas conducted.

Response 6
It is difficult to perform serotyping of the strains isolated from IPD cases in the other nine prefectures in Japan because of the lack of an IPD surveillance system.Therefore, we performed serotyping of the isolated strains based on the clinicians' request. We have highlighted this in the methods section.
"The samples from nine other prefectures were obtained from clinicians on request, because of the lack of active IPD surveillance system including bacterial analysis in the nine prefectures. " Also "S. pneumoniae is one of the (l)eading causes of invasivepneumococcal diseases (IPDs)" does not make sense as by definition it is the onlycause of invasive pneumococcal diseases.

Response 9
We thank you for your comment. We have revised the sentence per your comment. Line 173-175 I am not sure what is referred to in the BLAST search in the first ofthe two sentences across lines 175-177. The second sentence is followingthe methods description of comparing the cps among a subset of amplifiesand sequenced isolates. The first one appears to be comparing allisolates, although only a subset were sequenced. Or this may be areference to a BLAST search of isolates on public databases? It would begood to make this clear, including a statement in the methods toanticipate this aspect of the results. "The BLAST search resultsrevealed that the cps loci (except the abpA gene sequences) of all theserogroup 24 strains shared 100% identity. The cps loci (except the abpAgene sequences) of all the175 five tested strains of 24F ST 2572 and 24BST 2572 were identical."

Response 11
We thank you for your comment. We apologize for the confusion regarding the method for the molecular characterization of gene encoding capsular polysaccharides. We have revised the relevant sentences in the methods section.
"The original primers were created using primer design software (Primer-BLAST) based on the sequences available on GenBank, accession numbers CR931688 (serotype 24F) and CR931687 (serotype 24B), which are known sequences at the cpslocus of serogroup 24. The PCR products of the cpslocus were sequenced using the Sanger method. "(Page 3 Line 135-139) The abpAgene present in the cpslocus was analyzed in the 61 strains using PCR and DNA sequencing. Primers 8582F(5′-CAGC TGGA AAGT TAAT GGTTGGT-3′) and 9758R(5′-ACCA ATCA AACC AGAA GCTCCA-3′) were used in the PCR, following a published protocol [15]. Annotation of the determined gene sequences showed that the cpslocus configuration of all serogroup 24 ST2572 strains was the same as that of the gene used for reference (CR931688). Sequence comparison revealed some scattered single nucleotide polymorphisms (snps) at the cpslocus but no mutations common to strains 24F ST 2572 and 24B ST 2572 (except the abpAgene sequences). Therefore, we analyzed the abpAgene sequences of isolates 24F, 24B, and 24C that were isolated in this study. The PCR products were sequenced and the nucleotide sequence of abpAgene was determined. The relationship between the abpAgene present in the cpslocus and the ST/serotype combinations of the 61 strains was examined." Line 232: "serotyp0e" "serotype"

Response 12
We have fixed the mistake. We apologize for the oversight. (Page 5 Line 248)

Question 13
Discussion -There is some discussion of external publication as regardsthe increase in non-vaccine serotypes being widely observed in othercountries post vaccination, and some detail on ST-serotype distributionsin some other datasets. The emergence of serotype 24 C is set in theinternational context. However a fuller comparison of the dynamics ofserotype 24 generally in other countries post vaccination is missing andI think a gap. Subject to any journal space limitations expanding thisaspect of the introduction/ discussion would be useful.

Response 13
We thank you for your valuable comment. We have added the necessary description in the introduction section.
"Serogroup 24 includes serotypes 24F, 24A, 24B, and the newly identified 24C [7]. Serotype 24F as one of the most prevalent NVTs causing IPDs [8,9]. The prevalence of serotype 24 differs among countries. and with differentprevalence of serotypes among countries. A systematic review of serotype distribution of pediatric IPDs in the post-PCV era revealed that 24F prevalent in Europe and Western Pacific region, but not in North America [8]. " (Page 2 Line 52-55)

Question 14
The proposedmechanism for generation of variation is highly speculativebut is onlypresented as a "possibility" in line with this speculative and sketchynature.

Response 14
We agree with your comment. We have added a description in the main text.
"However, these proposed mechanisms underlying the variations have not been fully demonstrated. Considering the above hypothesis, a thorough investigation of the serogroup 24 strains isolated from sterile body sites and nasopharynx is necessary. Further studies are needed to clarify the precise mechanism. " Comments: Dear Dr Ishiwada Thank you for submitting your paper to Access Microbiology. Your manuscript has now been reviewed and I would like you to address their comments, in line with the provided reports at the end of this email. The reviewers and I agree that whilst your work on epidemiological and genetic characterisation of serogroup 24 pneumococcal isolates, provides a valuable addition to the existing literature and is of interest to readers of Access Microbiology. However some major changes have been suggested by the reviewers on the details of the methods provided and on the presentation/interpretation of the results that will improve the readability of the manuscript and the reproducibility fo the data. I would like you to consider, where appropriate, those comments and address them in a revision. Furthermore, given the open access policy of Access Microbiology, I would kindly ask to consider the possibility to make the data that support this study available on a public database. Comments: Methodologically this is mainly simple and clearly explained. My main methodological questions are about the sampling. The Chiba sample seems to be complete. I am less sure of the extent to which the wider survey of samples across Japan is representative or may be biased. This could be in terms of differential sampling across the time period, the selected clinicaldemographic population, and subtype. Here the description "samples from other prefectures were obtained from clinicians on request" does not give much idea. It would be good to be more explicit on this and the extent to which this may have biased the pattern across years, subtype or patient demography or clinical picture. If this is likely then features such as counts over time may be better restricted to the Chiba prefecture sample or at least for this to be visible in presentation and discussion of results across these dimensions. The other sampling issue is the overall timeframe. The title "Increase in prevalence of Streptococcus pneumoniae serogroup 24 in children upon introducing 13-valent pneumococcal conjugate vaccine in Japan" would lead me to expect as ample comparing afterwards with before. However the vaccination was introduced in 2010 (7 valent) and 2013 (13 valent) but the sample extends forward from 2015. At least for the 7 valent this was very late in terms of the main carriage group for most S. pneumoniae being your children with quite extensive coverage in place for this population before 2015. The rationale for this sampling period vs what might be expected and inferred from the title is not discussed or mentioned in limitations. The data summary statement notes that "No data was generated during this research." This seems to conflict with a description of methods to generate data and the data then presented in results as part of this research. There is also no statement on depositing of the data generated. Part of the usefulness of this research is for these data to be curated and accessible to other researchers and this should be assured and described. Presentation of results The paper generally presents data clearly. In comparing the sequences in table 2 the main findings to me look to be that (i) ST162 has many differences shared across all ST162 isolates and not present in the other STs., (ii) there is a lot of diversity within ST2572 serotype 24B, the recently emerged (or at least identified) serotype 24C is present across at least sequence types. I think that it would be useful to pull out these themes in the results descriptions and discussion. Also (Line 237 : "…point mutations were detected in the abpA sequences of all the 24B ST 2754 strains,…") sounds to me as though describing a range of mutations but Table 2 appears to show a single shared difference across 24B ST 5724 vs the reference sequence used in the paper. As above -ther emay be a reason to present the Chiba prefecture results as a population sample and other isolates not as a representative population sample subject to how this process of sampling more widely was conducted. MINOR DETAILS Line 55 : This serogroup has not targeted by …. This serogroup has not been targeted in … Line 77 : reading.. leading.. Also "S. pneumoniae is one of the (l)eading causes of invasive pneumococcal diseases (IPDs)" does not make sense as by definition it is the only cause of invasive pneumococcal diseases. Line 107: …various… …from various… Line 173-175 I am not sure what is referred to in the BLAST search in the first of the two sentences across lines 175-177. The second sentence is following the methods description of comparing the cps among a subset of amplifies and sequenced isolates.

Reviewer 2 recommendation and comments
The first one appears to be comparing all isolates, although only a subset were sequenced. Or this may be a reference to a BLAST search of isolates on public databases? It would be good to make this clear, including a statement in the methods to anticipate this aspect of the results. "The BLAST search results revealed that the cps loci (except the abpA gene sequences) of all the serogroup 24 strains shared 100% identity. The cps loci (except the abpA gene sequences) of all the175 five tested strains of 24F ST 2572 and 24B ST 2572 were identical." Line 232: "serotyp0e" "serotype" Discussion -There is some discussion of external publication as regards the increase in non-vaccine serotypes being widely observed in other countries post vaccination, and some detail on ST-serotype distributions in some other datasets. The emergence of serotype 24 C is set in the international context. However a fuller comparison of the dynamics of serotype 24 generally in other countries post vaccination is missing and I think a gap. Subject to any journal space limitations expanding this aspect of the introduction/ discussion would be useful. The proposed mechanism for generation of variation is highly speculative but is only presented as a "possibility" in line with this speculative and sketchy nature.

Please rate the manuscript for methodological rigour Satisfactory
Please rate the quality of the presentation and structure of the manuscript Good

Anonymous.
Date report received: 21 November 2022 Recommendation: Major Revision Comments: The authors present an epidemiological/capsular polysaccharide genetic analysis of serogroup 24 pneumococcal isolates from cases of invasive disease in Japan between 2015 and 2021. They demonstrate a switch in dominance of 24F to 24B, a loss of ST 2572 24F, the discovery of 24C for the first time in Japan and finally a potential clonal expansion of a capsular switch event involving 24B ST2754. Serogroup 24 is important in the context of pneumococcal disease and the authors are right to examine the epidemiology against a backdrop of PCV implementation. Methodological rigour, reproducibility and availability of underlying data The methods used are generally ok. However, there are several gaps in the methodology descriptions that would make reproduction difficult and therefore need to be addressed. 1.
The age distribution of cases is not clear. I would argue that 15-year-olds are better described as adolescents and not children and would urge the authors to reflect on this and alter. 2. For those unfamiliar with the study locations, it would be good to give a brief overview of the prefecture in terms of location and population, and the sites these isolates came from. 3.
It is not clear how many isolates came from other prefectures. Please clarify. 4. You should indicate how many pneumococcal isolates were available in total from which these 61 were taken. That would give an indication of the overall prevalence of serogroup 24 as a cause of IPD relative to other serotypes. 5. The descriptions of the methods need improving. For example, was it Columbia Blood Agar? If so, what was the manufacturer. How was optochin sensitivity assessed? Manufacturer of discs? 6.
You also need to give the PCR reaction conditions (primer concentrations, mastermix, thermocycler used and cycling conditions). This also applies to cps sequencing (where you should also supply the name of the sequencing instrument used and where it was done. 7. Line 174: Blast is mentioned here but there is no mention in the methods section. Presentation of results 1. Line 143-144. The optochin sensitivity and lytA PCRs do not confirm serogroup. 2. Line 144. Please can the authors clarify how in the methods it states that 61 serogroup isolates were acquired and here you state that it was 31, is this just a typo? 3. Figure 1. I do not have an issue with presenting counts however you should also present proportion as this figure does not illustrate whether overall IPD has remained constant over this period. Also, Y axis needs to be clearer. 4. The authors should consider a table of case demographics, or perhaps a figure showing when cases (by age) were isolated. 5.
Line 174-175. It's not clear why you tested all cps loci and then a subset of five. This requires clarifying. 6.
Please put the detail that all 24F ST2572 abpA were identical into Table 2. 7. Perhaps the information in Table 2 would be better communicated alongside a Figure. How the style and organization of the paper communicates and represents key findings The overall structure and organisation of the paper works for the data being presented. Literature analysis or discussion 1. I don't believe ST2754 has been previously associated with 24B and I think this potential capsule switch requires further discussion. 2.
Line 190: Whilst serogroup 24 might be rare in adults in Japan it nevertheless was reported in France (just as an example, Ouldali et al., Lancet ID, 2020). This should be reflected in the discussion. 3. Line 196: I'm not sure I follow here. You state that most isolates had specific STs which suggests some did not? Can you clarify please. 4.
Line 233-239: is the heterogeneity of abpA in 24B ST2754 common? I think this needs expanding as a discussion point. 5.
The 'limitations' section of the study requires expanding. AMR data would have been useful, and achievable. Whilst more challenging and costly the lack of genomic resolution of lineages, i.e., GPSCs, does make contextualisation difficult. Any other relevant comments Line 75: Gram should be capitalised Line 77: re-word, there are no other causes of IPD other than pneumococci Line 81: Specify that this is in adults Line 90: There is more to this reference than is given in this introduction. For the reader it would be useful to expand to highlight the GPSC narrative of these findings. Line 96-97: This implies that this has not been described previously, whereas it has. Perhaps rephrase to indicate this is a description of isolates from a specific geographic area. Line 118: Please cite the Jolley et al., 2018 paper in Wellcome Open Research for pubMLST. Line 207 (and elsewhere): please replace bacteria with pneumococci Line 232: typo with Serotype